Vary Substrate Phosphatase Enzyme Test
The data points on the graph present an increasing trend; however there is a large standard deviation.
There a a lot of data points within the 0.6 absorbence range which may show that there is some activity around that value.
Fig 2. Substrate Phosphate Enzyme Test
Excel Chart with Values
Fig 1. Values of Initial/Final Concentrations along with Standard Deviations
Week 13: Protein Purification/Characterization Images
Fig 3. Concentrated Elution 1 (Concentration increased as expected)
Fig 2. Concentration of Elution 2
Fig 1. Concentration of Elution 1
Week 12: Continuation With Protein
Proceeded to protein purification and characterization.
Lane 1: Skipped. Lane 2: PageRuler Ladder. Lane 3: Sample 0, Cell lysate before induction. Lane 4: Sample 1, Cell lysate after induction. Lane 5: Sample 3 Flow through. Lane 6: Wash. Lane 7: Elution 1 Lane 8: Elution 2
Sample 2 (Soluble Fraction) was lost so it was not included in the PAGE Gel.
. Week 11: Virtual
Top 10 Ligands from Virtual Screen. Highest fitness score is 84.23
Week 10: Protein Expression
Fig 1. Image of Gel before being stained
Day 3 completed
Materials & Methods for cloning completed
Week 9: Protein Expression
Day 2 started and completed Week 8: Virtual Screening
Virtual Screening on target started however waiting for run to start (error: need license)
Week 7: PCR Overlap Nanodrop
Nanodrop: concentration of cleaned PCR product via spectrophotometry; 260 ratio: 1.87; 280 ratio: 2.34
The graph shows that I have a very high concentration Week 6: Primer Overlap
Primer Overlap started on 10/7. Oligo mix created so PCR can be ran.
10/14 Primary PCR and Secondary PCR ran in gel in order to check if the process was working. The next step is to perform PCR squared.
Lane 1: Skipped; Lane 2: 100bp DNA Ladder; Lane 3: Primary PCR Reaction; Lane 4: Secondary PCR Reaction
This image shows the oligo mix was made correctly. The primary PCR product looks like a smear and the secondary PCR has a distinct band that corresponds to the size of the gene. It is safe to move on to the PCR squared.
Primer Design: Oligonucleotides for Histidine Phosphatase (Francisella Tularensis)
Blades were full for a couple of days and when the first run was checked, there was an error. So refresher was restarted. Results coming soon!
PCR
PCR was ran again after failing the 1st time.
Lane 1: Skipped, Lane 2: 100bp DNA Ladder, Lane 3: PCR pNIC-Bsa4 (Highest Conc. of pLIC), Lane 4: PCR pNIC-Bsa4 (Medium Conc. of pLIC), Lane 5: PCR pNIC-Bsa4 (Lowest Conc. of pLIC)
Lane 6 is supposed to be control for PCR. May have added extra sample into loading well. There are also three bands which is unusual. Week 4:PCR for pNIC-Bsa4 cloning off of Cloning Vector
Steps
1) Select primers:
VDS15 For: CA2 Human, pNIC-Bsa4 (2.5 uM)
VDS16 Rev: CA2 Human, pNIC-Bsa4 (2.5 uM)
2) Make master mix
3) Add master mix to each tube
4) Add DNA template to each tube (ID:4190, 383.1 ng/ul)
5) Add water
6) Add MgSO4
7) Keep on ice
8) Preheat PCR machine
9) Add Taq polymerase dilution (2ul+8ul Nanopure)
10) Run PCR machine
11) Make Agarose Gel
Lane 2: 100 bp DNA Ladder
With respect to the image above, it's clear to see that the experiment failed. This may have happened because of contamination or incorrect preparation of dilutions (1:10; 1:100; 1:1000).
Week 3: PyMol Results
Objective Examine three dimensional structure of a new enzyme
Background DHFR-TS from Trypanosoma cruzi is a bi-functional enzyme complex that carries out the role of dihydrofolate reductase and thymidylate synthase. T. cruzi is the pathogen responsible for Chagas disease (also called American trypanosomiasis), which causes approximately 50,000 deaths annually. The disease is endemic in South and Central America. In the chronic form, Chagas disease causes severe damage to the heart and other organs. There is no satisfactory treatment for chronic Chagas disease and no vaccine is available. Potentially, this target could be used to inhibit growth of the parasite.
9-15-11 2H2Q3:00pm This is the PDB identifier for the complex with the natural substrates. Make a PyMol image showing all of the components separately (each component should be selected individually and given a name). Display each chain distinctively. Show polar contacts between the protein and any substrates or cofactors.
Fig 1. Chain A of 2H2Q with substrate NAP (blue) shown as polar contacts to the active site indicated in (green) as sticks.
Fig 3. MTX (yellow) is shown as sticks inhibiting the 3CL9 active site indicated by the polar contacts within 5 Angstroms.
Fig 5. Pairwise comparison of the f 1U72 (yellow) and 3CL9 (green). Slight difference in the amino acid sequence in the active site is serine in 1U72 and lysine in 3CL9.
Fig 6. TMQ (yellow) is shown as sticks inhibiting the 3CL9 active site indicated by the polar contacts within 5 Angstroms. T. cruzi DHFR-TS is different than f MTX to human DHFR from 1U72. The aromatic rings of TMQ are hydrophobic . Active site is red color.
Fig 7. Image zoomed out to show four chains with different colors. Active site is black in each chain. TMQ inhibitors are shown as sticks. Polar contacts also included (light red).
Conclusion: It appears as if 1U72 and 3CL9 are similar when with respect to alignment scores. The similarities are that the active sites and the orientations of the MTX are extremely similar while some differences are that just a few amino acids are different in the active site. It’s also important to note that MTX is also the inhibitor for both of the proteins. Polar contacts help determine the binding affinity for different active sites. Essentially MTX has a stronger binding affinity for the active site of DHFR than TMQ to DHFR-TS.
Week 2: RE Digest
Day 1
1) Digestive Reactions
Eco RI
PvuII
EcoRI+PvuII (double digestion in parallel)
2) Digestion in 1.7 ml centrifuge tube
3) Incubate at 37 degrees Celsius for 1-2 hours
4) Heat block for 20 min
Day 2
1) Make Agarose Gel
2) Run gel for about 40-45 min
Week 1: PCR Protocol
Day 1
1)Make Template Dilutions
2) Make Master Mix
3) Add Master Mix to each tube
4) Add template to each tube
5) Place on ice
6)Preheat PCR machine
7) Add Taq polymerase
8) Run PCR machine
Day 2
1) Make Aragose Gel
2) Run Gel for about 40 min
T.J. - what is your template for this PCR below? Also, what is special about Sample D? How is Sample A,B,C different? - Dr. B
100 bp DNA Ladder Reference
Aragose Gel: 9/7/11
Used 426.3 ng/micro liter of pGBR22 template. M13 forward and reverse primers (primer mix) were used. There is a clear distinction of where the band is. Shows up in the secondary lanes (3-5) which prove that the PCR worked. The best lane to chose from would be lanes 4 (sample C) and 5 (sample D) simply because of less fragmentation. The ladder on the gel also shows up very good as well.
Lane 1: 100bp DNA ladder + Blue Juice (7.0ul) Lane 2: Sample A (12.0ul) Lane 3: Sample B (12.0ul) Lane 4: Sample C (12.0ul) Lane 5: Sample D (12.0ul)
TJ's Research
Week 14: Assay Tests
Vary Substrate Phosphatase Enzyme Test
The data points on the graph present an increasing trend; however there is a large standard deviation.
There a a lot of data points within the 0.6 absorbence range which may show that there is some activity around that value.
Excel Chart with Values
Week 13: Protein Purification/Characterization Images
Week 12: Continuation With Protein
Proceeded to protein purification and characterization.
Sample 2 (Soluble Fraction) was lost so it was not included in the PAGE Gel.
.
Week 11: Virtual
Week 10: Protein Expression
Day 3 completed
Materials & Methods for cloning completed
Week 9: Protein Expression
Day 2 started and completed
Week 8: Virtual Screening
Virtual Screening on target started however waiting for run to start (error: need license)
Week 7: PCR Overlap Nanodrop
The graph shows that I have a very high concentration
Week 6: Primer Overlap
Primer Overlap started on 10/7. Oligo mix created so PCR can be ran.
10/14 Primary PCR and Secondary PCR ran in gel in order to check if the process was working. The next step is to perform PCR squared.
This image shows the oligo mix was made correctly. The primary PCR product looks like a smear and the secondary PCR has a distinct band that corresponds to the size of the gene. It is safe to move on to the PCR squared.
Primer Design:
Oligonucleotides for Histidine Phosphatase (Francisella Tularensis)
26 oligonucleotides need to be synthesized
----------------------------------------------------------------
1 ATGAAGAAAATCTTCGTTTCTTTCACGCTGCTGTTCTTCCTGATCCCTGTTGGTTACTC 59
2 CCGTGACGGGTGATCATAGACACGAAAATCAGTTTAGAAGAGTAACCAACAGGGATCAGG 60
3 CTATGATCACCCGTCACGGTGACCGTGCACCGTTCGCCAACATCGAAAACGCGAACTATT 60
4 GCCAATCGGGGTCAGTTCAGACAGTTCGGTACCCCAAGAATAGTTCGCGTTTTCGATGTT 60
5 GAACTGACCCCGATTGGCATGAACCAGGAATACAACCTGGGTCTCCAGCTGCGTAAACGT 60
6 GTCAACGTAATGTTCCGGGAGCAGACCGAATTTATCGATGTAACGTTTACGCAGCTGGAG 60
7 TCCCGGAACATTACGTTGACCAGTCTATCTACGTTCTGTCTTCTCACACCAACCGTACCG 60
8 CAGCAGGGTACAGACCCATCAGCAGAGACTGGGCGCTAACTACGGTACGGTTGGTGTGAG 60
9 TGGGTCTGTACCCTGCTGGCACCGGTCCGCTGATCGGTGACGGCGACCCAGCGATCAAAG 60
10 AGTCCGCAGACAGGGTCATAATCGGGATCGGCTGGAAACGATCTTTGATCGCTGGGTCGC 60
11 GACCCTGTCTGCGGACTCTCGTCTGATCCAGTTCCCGTACGAACAGTACCTCGCGGTTCT 60
12 GTTTTGTTTTGCCATTCCGGAGAATTGTAAACATACTTTTTGAGAACCGCGAGGTACTGT 60
13 TCCGGAATGGCAAAACAAAACCAAAGAAGCGGCTCCGAACTTTGCGAAATGGCAGCAGAT 60
14 CGGTGATAACGTCGTTCAGGCCAGAAATACGATTACCCAGGATCTGCTGCCATTTCGCAA 60
15 CCTGAACGACGTTATCACCGTTGGTGATGTCCTGATTGTTGCGCAGGCGCACGGTAAGCC 60
16 CGATGATTTGGTCCGCGTCTTCTTGAGAGAGACCTTTCGGCAGAGGCTTACCGTGCGCCT 60
17 ACGCGGACCAAATCATCGCGCTCACCGACTGGGGTCTGGCACAGCAATTTAAGTCTCAGA 60
18 GCGATTGGTGAGTTTACCACCCATGATGTAAGAAACTTTCTGAGACTTAAATTGCTGTGC 60
19 GTGGTAAACTCACCAATCGCATGATTGAAGACCTGAACAACGCGGTTAATGGTAAGTCTA 60
20 GAGTCAGGTCGTGACCAGAGTAGTAGGTCATTTTGTACTTAGACTTACCATTAACCGCGT 60
21 TCTGGTCACGACCTGACTCTGCTCGAAGTTATGGGCACCCTGGGTGTTCCGCTCGATACC 60
22 TTTGTACAGTTCCATTTCCAGGTTAGACGCGTAACCCGGTGCGGTATCGAGCGGAACACC 60
23 CCTGGAAATGGAACTGTACAAAGATGGTGATATCTACACGGTTAAACTGCGTTACAACGG 60
24 GAGTTGTTTTTGTCCATAATAGGGAGTTTGACGTATTTACCGTTGTAACGCAGTTTAACC 60
25 TCCCTATTATGGACAAAAACAACTCTTGCTCTCTGGACGCGCTGAATAAGTATATGCAAT 60
26 TTACTTCTGGAATTTTTCGTTAATAGATTGCATATACTTATTCAGCGCG 49
Week 5: Virtual Refresher
Virtual Screening (Started 1st Run)
Blades were full for a couple of days and when the first run was checked, there was an error. So refresher was restarted. Results coming soon!
PCR
PCR was ran again after failing the 1st time.
Lane 6 is supposed to be control for PCR. May have added extra sample into loading well. There are also three bands which is unusual.
Week 4: PCR for pNIC-Bsa4 cloning off of Cloning Vector
Steps
1) Select primers:
VDS15 For: CA2 Human, pNIC-Bsa4 (2.5 uM)
VDS16 Rev: CA2 Human, pNIC-Bsa4 (2.5 uM)
2) Make master mix
3) Add master mix to each tube
4) Add DNA template to each tube (ID:4190, 383.1 ng/ul)
5) Add water
6) Add MgSO4
7) Keep on ice
8) Preheat PCR machine
9) Add Taq polymerase dilution (2ul+8ul Nanopure)
10) Run PCR machine
11) Make Agarose Gel
With respect to the image above, it's clear to see that the experiment failed. This may have happened because of contamination or incorrect preparation of dilutions (1:10; 1:100; 1:1000).
Week 3: PyMol Results
Objective
Examine three dimensional structure of a new enzyme
Background
DHFR-TS from Trypanosoma cruzi is a bi-functional enzyme complex that carries out the role of dihydrofolate reductase and thymidylate synthase. T. cruzi is the pathogen responsible for Chagas disease (also called American trypanosomiasis), which causes approximately 50,000 deaths annually. The disease is endemic in South and Central America. In the chronic form, Chagas disease causes severe damage to the heart and other organs. There is no satisfactory treatment for chronic Chagas disease and no vaccine is available. Potentially, this target could be used to inhibit growth of the parasite.
9-15-11 2H2Q 3:00pm
This is the PDB identifier for the complex with the natural substrates. Make a PyMol image showing all of the components separately (each component should be selected individually and given a name). Display each chain distinctively. Show polar contacts between the protein and any substrates or cofactors.
Conclusion:
It appears as if 1U72 and 3CL9 are similar when with respect to alignment scores. The similarities are that the active sites and the orientations of the MTX are extremely similar while some differences are that just a few amino acids are different in the active site. It’s also important to note that MTX is also the inhibitor for both of the proteins. Polar contacts help determine the binding affinity for different active sites. Essentially MTX has a stronger binding affinity for the active site of DHFR than TMQ to DHFR-TS.
Week 2: RE Digest
Day 1
1) Digestive Reactions
- Eco RI
- PvuII
- EcoRI+PvuII (double digestion in parallel)
2) Digestion in 1.7 ml centrifuge tube3) Incubate at 37 degrees Celsius for 1-2 hours
4) Heat block for 20 min
Day 2
1) Make Agarose Gel
2) Run gel for about 40-45 min
Week 1: PCR Protocol
Day 1
1)Make Template Dilutions
2) Make Master Mix
3) Add Master Mix to each tube
4) Add template to each tube
5) Place on ice
6)Preheat PCR machine
7) Add Taq polymerase
8) Run PCR machine
Day 2
1) Make Aragose Gel
2) Run Gel for about 40 min
T.J. - what is your template for this PCR below? Also, what is special about Sample D? How is Sample A,B,C different? - Dr. B
Aragose Gel: 9/7/11
Used 426.3 ng/micro liter of pGBR22 template. M13 forward and reverse primers (primer mix) were used. There is a clear distinction of where the band is. Shows up in the secondary lanes (3-5) which prove that the PCR worked. The best lane to chose from would be lanes 4 (sample C) and 5 (sample D) simply because of less fragmentation. The ladder on the gel also shows up very good as well.
Also show a clear band within lanes 3-5.